Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membrane-bound receptors

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC α along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC ?. In contrast, thrombin had a smaller effect on nuclear translocation of PKC α and did not influence PKC ?, but instead induced a rapid nuclear translocation of PKC ζ. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC α and ? within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms α, δ,? and ζ in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC α and ? to the perinuclear region and into the nucleus, while PKC δ and ζ showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC α and ? was reduced to or below control values. At 8 h, increased nuclear expression of isoforms α,? and ζ was observed, while isoform δ was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.     

Hypofluorescent spots in indocyanine green angiography of peau d‘orange and fundus

Purpose. Hypofluorescent spots were seen inindocyanine green (ICG) angiography of peau dorangefundus in eyes with angioid streaks. Origin of the hypofluorescentspots were examined with attention to their correlationwith a peau dorange appearance of the central fundususing a computer-assisted image comparison system. Methods. ICG angiography was performed in 5 patientshaving peau dorange appearance of fundus using ascanning laser ophthalmoscope (SLO) and a digitalvideo-fundus camera. The same central fundus areas corresponding to hypofluorescent spots in an ICGangiogram were then digitally identified in afluorescein angiogram and in a red-free picture in all10 eyes of the 5 patients. Monochromatic lightobservation was also performed with a dark fieldobservation using a SLO to see subretinal orintrachoroidal pigment clumping. Results. In no patient, the areas identified withhypofluorescent spots did show relevant changes ina fluorescein angiogram or a red-free picture. SLOexamination revealed not perfusion defect at the sameareas. The dark field observation showed no pigmentclumping at the peripapillary and papillomacularbundle regions where hypofluorescent spots were seen.Conclusions: Hypofluorescent spots seen in ICGangiograms did not show exact consistency with peau dorange changes in their location and shape. Perfusion defects or blocking by pigments were not acause of hypofluorescent spots. The scatteredhypofluorescent spots were considered to be relevantwith irregular affinity of the fundus to ICG dye.     

Ca2+ activation of hSlo K+ channel is suppressed by N-terminal GFP tag

The human slow poke (hSlo) K+ channel was tagged with GFP (green fluorescent protein) at the N-terminus of its alpha-subunit. The fusion protein was expressed transiently in HEK293 cells; it formed functional voltage-gated channels as shown by whole cell patch-clamp measurements. However, the tag lowered the voltage dependence of gating and it suppressed the typical left-shift of gating by intracellular binding of Ca2+. The location of the GFP-tagged N-terminus was confirmed to be on the extracellular side by application of a monoclonal antibody to nonpermeabilized cells. Structural interpretations of the effects are discussed.     

Indocyanine green guided laser photocoagulation in patients with occult choroidal neovascularisation

AIMS: To determine whether indocyanine green (ICG) guided laser photocoagulation of occult choroidal neovascularisations (OCNV) is beneficial for patients with occult choroidal neovascularisation secondary to age related macular degeneration (AMD). METHODS: A prospective pilot study was performed in 21 eyes with OCNV secondary to AMD that could be identified extrafoveolarly or juxtafoveolarly in an early ICG angiographic study. Laser photocoagulation was applied to the neovascular membrane identified in the early ICG angiographic study. RESULTS: Visual acuity ranged from 20/400 to 20/20 (logMAR 0.54 (SD 0.29) before and hand movements and 20/30 (logMAR 0.81 (0.69)) at the last follow up after laser photocoagulation. During the follow up (30 (13) months) vision improved in four eyes (two lines), in seven eyes the initial visual acuity could be stabilised (two lines), in five eyes vision dropped moderately (three to five lines), and in five eyes vision decreased severely (six or more lines). Recurrences (seven patients) or persistent CNV (six patients) was observed in 13 patients. CONCLUSION: This preliminary study of ICG guided laser photocoagulation of occult extrafoveal and juxtafoveal choroidal neovascularisations suggests that this technique may improve the visual prognosis of these patients. Further prospective controlled studies are necessary to confirm these data.     

Prognostic value of galactose elimination capacity,aminopyrine breath test,and ICG clearance in patients with cirrhosis

Seventy-eight patients with cirrhosis were prospectively followed for up to 20 months, on the average. At entry into the study, galactose elimination capacity, aminopyrine breath test, and ICG clearance were measured. At the end of the study, 27 patients had died. Univariate analysis using the Kaplan-Meier method showed that both quantitative liver function tests (galactose elimination capacity:P<0.025; aminopyrine breath test:P<0.001; ICG clearance:P<0.005) and common clinical and biochemical data (encephalopathy:P<0.001; ascites:P<0.001; serum bilirubin:P<0.005; serum albumin:P<0.001; prothrombin index:P<0.05) were significant predictors of survival. To investigate whether quantitative liver function tests could contribute to a better definition of the prognosis, once Pugh score had already been taken into account, a multiple regression analysis according to the Cox model was performed. Pugh score and galactose elimination capacity resulted in the only independent prognostic covariates. From them a prognostic index was calculated, and the model was validated in an additional sample of 70 patients investigated according to the same protocol. The contribution GEC gave to the assessment of overall prognosis over that obtained using the Pugh score was slight, as estimated by the statistical parameters of the Cox's model, but was significant as assessed by a ROC curve analysis (P=0.05). These data show that all quantitative liver function tests were predictors of survival in cirrhosis, and that the galactose elimination capacity added some new prognostic information to those already available using the Child-Turcotte-Pugh classification.This study was supported in part by a grant from the Italian Ministry of Education (National Project Liver Cirrhosis). Part of this study was presented at the 22nd Meeting of the European Society for Clinical Investigation, Graz, Austria, April 20–23, 1988.     

单侧渗出型老年性黄斑变性对侧眼的吲哚青绿血管造影特征

目的 探讨单侧渗出型老年性黄斑变性(AMD)对侧眼吲哚青绿血管造影(ICGA)特征。方法 分析34例经ICGA确诊的单眼渗出型AMD患者对侧34只眼的荧光素眼底血管造影(FFA)及ICGA图像资料。结果 单眼AMD患者对侧眼分别有中晚期后极部簇状强荧光点、强荧光斑、斑片状强弱相间的荧光图像，以及脉络膜灌注不良等几种异常荧光形态。玻璃膜疣在ICGA和FFA上显示强荧光、弱荧少．和正常荧光3种表现；玻璃膜疣在FFA呈强荧光，ICGA一直为弱荧光有18只眼。ICGA中晚期后极部见簇状分布的强荧光点5只眼；中晚期出现1个或多个强荧光斑6只眼；脉络膜灌注不良7只眼。结论 一眼发生渗出型AMD，ICGA检查有助于发现对侧眼是否有病变、病变程度，以及判断预后。     

MADP-1基因对前列腺癌细胞系DU145体外生长的影响

[目的]探讨新基因MADP-1对雄激素非依赖性前列腺癌细胞系DU145增殖的影响,了解其功能.[方法]采用脂质体和绿色荧光蛋白基因标记的质粒载体pIRES-hrGFP-1α,将MADP-1基因导入前列腺癌细胞系DU145中.应用荧光显微镜、流式细胞仪分析和分选术及细胞生长曲线动态观察MADP-1基因对DU145细胞系的影响.[结果]转染细胞在荧光显微镜下显示绿色荧光.转染MADP-1基因组细胞生长加速,与转染空载体组及对照组相比,有显著差异(P＜0.05).[结论]新基因MADP-1在体外具有促进前列腺癌细胞系DU145增殖的作用.     

老年性黄斑变性脉络膜新生血管的组织病理学特征

目的 观察老年性黄斑变性（AMD）脉络膜新生血管(CNV) 吲哚青绿血管造影(ICGA) 表现与其组织病理学变化的关系。 方法 ICGA检查确诊的AMD患者21 例21只眼，根据ICGA检查结果将CNV分成活动期、消退期、静止期，通过玻璃体切割手术取出CNV，光学显微镜、透射电子显微镜观察不同期CNV的组织病理学特征。 结果 活动期CNV9只眼，组织病理学特征为大量CNV，无色素性细胞和纤维组织或有少量色素性细胞将其部分包裹；消退期CNV9只眼，组织病理学特征为CNV较活动期减少，有较多色素性细胞、少量的纤维组织；静止期CNV3只眼，组织病理学特征为大量的纤维组织，无CNV或有极少CNV，无色素性细胞。 结论 渗出型AMD患者CNV的组织病理学特征与ICGA表现具有一定的相关性。 （中华眼底病杂志，2004，20：71-74）     

肝部分切除术对大鼠门静脉途径肝细胞转基因效率的影响

目的 :探讨肝部分切除术对大鼠肝细胞门静脉途径转基因效率的影响。方法 :Wistar大鼠分 4组 ,每组 5只 :A1组 :30 %切肝并于门静脉注射脂质体—质粒 DNA混合物 (lipofectamine- p EGFP- N1DNA complexes,lipoplexes) ;A2组 :30 %切肝并于门静脉注射生理盐水 (NS) ;B1组 :70 %切肝并于门静脉注射 lipoplexes;B2组 :70 %切肝并于门静脉注射 NS。转染后 15 d均以胶原酶消化、Percoll液梯度离心法获取纯化肝细胞悬液 ;以 NS组为对照并以 GFP为荧光标记物 ,用流式细胞仪 (flow cytometry,FCM)分析各实验组肝细胞的转染率。结果 :A1组和 B1组的平均转染率分别为 :(1.12± 0 .5 0 ) %、(3.91± 1.6 8) % ;B1组明显高于 A1组 (P<0 .0 5 )。结论 :与 30 %肝部分切除术相比 ,70%切肝可显著提高大鼠肝细胞门静脉途径转基因的效率     

不同转染方法及GFP表达载体对小鼠胚胎干细胞转染效率的比较

目的对比不同的转染方法及绿色荧光蛋白(GFP)表达载体对小鼠胚胎干细胞(ESC)的转染效率,为进一步研究ESC及衍生细胞移植示踪提供工具。方法用阳离子脂质体转染小鼠胚胎干细胞系ES-D3,对比贴壁细胞转染法、悬浮细胞转染法及不同载体pCX-EGFP、p-EGFP-C1和pIRES-hrGFP的转染效率。结果悬浮法和贴壁法转染效率分别为(90.0±4.1)%,(70.0±2.3)%,两组比较差异有显著性意义(P<0.05)。3种载体转染效率分别为pCX-EG-FP(90.0±4.1)%,pIRES-hrGFP(32.0±6.0)%,p-EGFP-C1(4.0±0.4)%。3组间两两比较差异均有显著性意义(P<0.05)。结论悬浮转染法转染pCX-EGFP可获得较高的转染效率。